Application of the FAIMS Pro Duo Interface for Selective Detection of Lower Abundance Lipid Classes at Analytical Flow Rates

Importance of the topic

The selective detection of low-abundance lipid classes in complex biological samples remains a critical challenge in untargeted lipidomics. Field-asymmetric ion mobility spectrometry (FAIMS) adds a gas-phase separation dimension upstream of mass analysis, reducing chemical noise, improving signal-to-noise ratios and enabling more confident identification of minor lipid species at analytical (high) flow rates.

Objectives and study overview

This work aimed to demonstrate the application of the Thermo Scientific FAIMS Pro Duo interface, coupled to a Vanquish UHPLC and Orbitrap ID-X Tribrid mass spectrometer, for selective enrichment and detection of lower-abundance lipid classes in untargeted LC-MS workflows. Lipid class standards and complex extracts from liver, heart, soy and brain were analyzed at 260 µL/min to evaluate class-specific compensation voltages (CVs) and improvements in coverage.

Methodology and Instrumentation

Chromatographic separation employed a Thermo Scientific Accucore C30 column (2.1 × 150 mm, 2.6 µm) at 45 °C using a water/acetonitrile (60:40) to isopropanol/acetonitrile (90:10) gradient with 10 mM ammonium formate and 0.1% formic acid. MS data were acquired in positive and negative ESI modes with and without FAIMS. Key instrumentation and software:

• Thermo Scientific Vanquish UHPLC system
• Thermo Scientific FAIMS Pro Duo interface
• Thermo Scientific Orbitrap ID-X Tribrid mass spectrometer
• Thermo Scientific Freestyle 1.8 SP2 software
• LipidSearch 4.2 for lipid annotation

Main results and discussion

Optimization using SPLASH LipidoMix standards (CV scans from –60 to +20 V in positive mode and –20 to +60 V in negative mode) revealed distinct CV optima for each lipid class. Analytical flow rates (260 µL/min) produced sharper CV separation than lower flows. Single-CV LC-MS runs on complex extracts achieved:

• Up to several-fold increases in signal-to-noise compared to no FAIMS
• Selective gas-phase enrichment, enabling resolution of isobaric pairs (e.g. PS(36:2)+Na+ vs PC(38:4)+H+)
• Higher confidence in class-specific MS/MS spectra
• Expanded lipid annotation counts across brain, heart, liver and soy matrices

Benefits and practical applications

The FAIMS Pro Duo interface at analytical flow rates:

• Reduces chemical background and matrix interferences
• Enhances detection limits for low-abundance lipid classes
• Facilitates separation of isobaric and coeluting species without extended chromatography
• Streamlines untargeted lipidomics workflows in research and QA/QC labs

Future trends and opportunities

Dynamic CV switching during single runs may broaden coverage of diverse lipid classes. Integration with advanced data-analysis tools and higher-resolution MS platforms could further improve selectivity. Expansion of FAIMS-based gas-phase fractionation to other biomolecular classes and multidimensional separations is anticipated.

Conclusion

This study demonstrates that the FAIMS Pro Duo interface, when operated at analytical LC flow rates, provides effective gas-phase fractionation to selectively enrich low-abundance lipid classes, improve signal-to-noise, resolve isobaric interferences and enhance annotation confidence in untargeted lipidomics.

Reference

1. Purves R.W.; Prasad S.; Belford M. et al. J. Am. Soc. Mass Spectrom. 2017, 28, 525–538.
2. Wei M.S.; Kemperman R.H.J.; Yost R.A. J. Am. Soc. Mass Spectrom. 2019, 30, 731–742.